二苯乙烯苷等药物对阿尔茨海默病APP可变剪接的调节及干预作用

发布时间：2018-04-19 18:22

本文选题：二苯乙烯苷 + 淀粉样前体蛋白　；参考：《首都医科大学》2017年博士论文

【摘要】：第一部分:二苯乙烯苷对APP可变剪接的调节和干预作用目的:β淀粉样蛋白(Aβ)聚集形成的老年斑是阿尔茨海默病(AD)的重要病理特征之一,由淀粉样前体蛋白(APP)经成淀粉途径多次水解形成。人APP基因表达受可变剪接调控,人脑内有三种APP可变剪接异构体产物,分别是APP770,APP751和APP695。APP外显子7编码类似Kunitz蛋白酶抑制子功能的结构域(Kunitz protease inhibitor domain,KPI),根据这些APP可变剪接异构体是否表达外显子7,可分为APP-KPI+和APP-KPI-两种类型。据报道,APP-KPI+在脑内的表达水平与Aβ的产生呈正相关。糖原合酶3β(Glycogen synthase kinase-3β(11)GSK3β)是脑内和阿尔茨海默病(Alzheimer’s disease,AD)密切相关的重要激酶之一,它的活性在AD脑内增加,是AD治疗中的一个潜在的重要靶点。我们的前期研究发现,何首乌主要成分二苯乙烯苷(tetrahydroxystilbene glucoside,TSG)能够减少APP转基因小鼠脑内Ab含量和淀粉样斑块数量,改善学习记忆功能,但TSG对APP可变剪接和GSK3β的影响尚不清楚。本课题的目的是从分子、细胞和动物整体水平系统研究GSK3β是否及如何通过磷酸化剪接因子从而调节APP外显子7的可变剪接,探索AD早期病变机制,探讨TSG对AD病理的影响,从而为临床治疗AD的新药创制提供分子理论依据。方法:(1)构建包含APP外显子7、8可变剪接的微型基因(mini-gene)。设计针对外显子6-9以及中间部分内含子的各段引物,进行PCR,然后将扩增片段插入真核表达载体PCI-neo,转染至HEK-293FT细胞系内进行剪接异构体表达产物检测。人胚胎肾细胞(HEK-293FT)、小鼠神经母细胞瘤细胞(Neuro-2a,N2a)内转染APP mini-gene(包含了外显子6-9,部分内含子6、7、8)来模拟APP外显子7、8在体内的剪接。(2)在人神经母细胞瘤细胞(SH-SY5Y)和N2a细胞系内检测APP微型基因的表达。针对APP770,APP751,APP695和总的APP设计特异的RT-PCR和荧光实时定量PCR(quantitative PCR,qPCR)引物,用普通PCR或荧光实时定量PCR(qPCR)测定APP各剪接异构体或APP-KPI+的表达量。在N2a细胞内过表达GSK3β或下调其表达,用RT-PCR和qPCR观察GSK3β对APP可变剪接的影响。(3)在HEK-293FT细胞内共表达剪接因子ASF(alternative splicing factor)和激酶GSK3β,用HA抗体免疫共沉淀ASF,GSK3β抗体检测ASF与GSK3β之间是否存在相互作用。此外,将HA-ASF、Myc-GSK3β转染至HEK-293FT细胞,然后将ASF免疫沉淀,用磷酸化特异性抗体检测免疫沉淀的ASF被磷酸化的程度。将ASF、GSK3β单独或者共转染至He La细胞中,用荧光二抗进行染色,观察ASF和GSK3β的细胞定位。(4)对SD(Sprague Dawley)大鼠进行侧脑室注射胰岛素(Insulin),6小时后行RT-PCR和Western blot检测各信号分子表达与活性,检测PI3K-AKT通路,观察APP-KPI+水平是否随之变化。APP/PS1转基因鼠TSG(50mg/kg/day)灌胃处理12个月,用Western blot或qPCR观察鼠脑内相关蛋白在m RNA和蛋白水平发生的改变。结果:(1)APP mini-gene在HEK-293FT和N2a细胞系内进行表达,RT-PCR结果可见三个条带,经测序分别是APP770,APP751和APP695。其中APP-KPI+(770和751)表达量较之APP-KPI-(695)丰富,因此该微型基因是研究APP-KPI+的理想工具。过表达GSK-3β促进APP微型基因产物APP-KPI+在N2a细胞内的表达,si GSK3β则抑制APP-KPI+表达量;不同浓度GSK3β抑制剂Li Cl处理SH-SY5Y细胞,内源性APP-KPI表达量随Li Cl浓度增高而降低。(2)各种剪接因子分别和APP微型基因共转染HEK-293FT细胞,其中ASF对APP-KPI+的表达影响最大,是最为重要的APP可变剪接调控因子。GSK3β可以被ASF免疫共沉淀,存在生理上的相互作用,并且免疫共定位实验显示ASF与GSK3β有很好的细胞内共定位。激酶GSK3β可增加细胞内剪接因子ASF丝氨酸磷酸化水平。然而,共表达ASF和GSK3β的细胞如果用TSG预处理48 h,ASF上丝氨酸磷酸化水平则下降。(3)TSG抑制HEK-293FT和SH-SY5Y细胞内APP-KPI+的表达量,同时发现SH-SY5Y细胞内源性AKT-GSK3β信号通路可被TSG激活,AKT和GSK3β的磷酸化水平显著增加,呈现浓度依赖性,而AKT和GSK3β本身表达量无明显变化。大鼠侧脑室注射PI3K-AKT-GSK3β信号通路激活剂Insulin后6 h,检测到AKT-GSK3β通路被激活。同时,APP-KPI+的表达水平与对照组相比下调P0.05。(4)在体内实验中,5月龄APP/PS1转基因小鼠灌胃给药TSG(50mg/kg/day)12个月后,脑内AKT和GSK3β磷酸化水平增加,APP-KPI+表达水平降低。结论:本实验成功构建了研究APP-KPI表达的微型基因。TSG可以激活神经细胞和APP/PS1转基因鼠脑内的AKT-GSK3β通路;GSK3β可磷酸化剪接因子ASF,从而抑制其促进APP-KPI+生成的能力;在体内长期喂食TSG可降低APP-KPI+表达,从而预防Ab沉积。结果提示,TSG可能通过激活AKT-GSK3β信号通路而调节APP可变剪接,从而改善Ab病理变化。第二部分:EGCG对Dyrk1A调节APP可变剪接的影响目的:AD的病理特征之一是由Ab沉积形成的老年斑。本室前期研究发现双特异性酪氨酸磷酸激酶1A(Dual specificity tyrosine-phosphorylation-regulated kinase1A,Dyrk1A)是脑内重要的蛋白激酶,其活性在AD脑内升高,同时Dyrk1A是一个潜在的基因可变剪接调节因子,已有的研究表明它介导了微管相关蛋白tau基因外显子10的可变剪接调控,但尚未有报道Dyrk1A对APP可变剪接的研究。表没食子儿茶素-3-没食子酸酯(epigallocatechin-gallate,EGCG)属绿茶天然提取物,毒性小。本研究的目的是研究Dyrk1A对APP微型基因和内源性APP基因可变剪接的影响;通过细胞内和整体动物抑制Dyrk A活性,研究APP-KPI的表达变化和Ts65Dn模式小鼠的学习记忆行为学改变。方法:(1)转染Dyrk1A真核表达质粒或无酶活性突变体(dominant negative)Dyrk1ADN至N2a细胞中,Real-time PCR检测Dyrk1A对APP-KPI+表达量的影响。(2)培养SH-SY5Y细胞至密度80%左右,将不同剂量的Dyrk1A抑制剂骆驼蓬减(Harmine)加入到DMEM/F12基础培养液中,24h后收取细胞提取细胞总RNA,用RT-PCR分析APP可变剪接变化。构建APP770表达质粒,转染至HEK-293FT细胞,用Western blot检测APP-KPI+表达改变,研究Dyrk1A抑制剂对APP-KPI+表达的影响。(3)抑制Dyrk1A活性对Ts65Dn模式鼠的行为学改变及分子生物学影响:从出生后即开始给予小鼠含Dyrk1A抑制剂EGCG的饮食,2-3mg/day。喂食3个月后观察喂药组和对照组小鼠脑内APP可变剪接的变化和学习记忆行为学改变。结果:(1)转染Dyrk1A和无活性的Dyrk1A至N2a细胞,发现Dyrk1A明显促进APP-KPI表达;而过表达无活性的Dyrk1A突变体,上述变化无显著差异。(2)不同浓度Dyrk1A抑制剂Harmine作用于SH-SY5Y细胞,能够抑制APP可变剪接,其表达产物APP-KPI+表达呈现Harmine浓度依赖性降低。(3)转染Dyrk1A各种缺失突变体至SH-SY5Y细胞中,观察到缺失C-端部分序列的Dyrk1A突变体与全长相比,不同程度促进APP-KPI表达。RT-PCR和Western blot统计分析结果均提示Dyrk1A的缺失突变体对APP可变剪接较之野生型影响更大。(4)行为学变化:Ts65Dn模式鼠和正常对照鼠相比,空间学习记忆能力显著降低。EGCG能够减低Ts65Dn小鼠脑内APP-KPI+水平,同时受损的空间学习记忆能力有所改善。结论:Dyrk1A调节APP可变剪接,过表达Dyrk1A促进APP-KPI+生成,抑制它的活性则APP-KPI+表达量降低。给予Ts65Dn模式鼠EGCG饮食能够抑制Dyrk1A活性,降低脑内APP-KPI表达量,改善学习记忆行为学变化。
[Abstract]:Part 1: two the regulation and intervention of two styrene glucoside on variable splicing: the accumulation of beta amyloid protein (A beta) is one of the important pathological features of Alzheimer's disease (AD), which is hydrolyzed by amyloid precursor protein (APP) through the starch pathway. Human APP gene expression is regulated by variable splicing and three in the human brain APP variable splice isomer products, APP770, APP751 and APP695.APP exon 7, encode the domain of the function of the Kunitz protease inhibitor (Kunitz protease inhibitor domain, KPI). According to whether these APP variable splice isomers express exon 7, they can be divided into two types: APP-KPI+ and two types. The expression level is positively related to the production of A beta. The glycogen synthase 3 beta (Glycogen synthase kinase-3 beta (11) GSK3 beta) is one of the important kinases closely related to the Alzheimer 's disease (AD) in the brain. Its activity is increased in the AD brain. It is a potential important target in AD treatment. Tetrahydroxystilbene glucoside (tetrahydroxystilbene glucoside, TSG) can reduce the Ab content and the number of amyloid plaques in the brain of APP transgenic mice and improve the learning and memory function. However, the effect of TSG on APP variable splicing and GSK3 beta is not clear. The purpose of this topic is to study the GSK3 beta from the molecular, cell and animal level system. And how to adjust the variable splicing of APP exon 7 by phosphorylated splicing factors, explore the early pathological mechanism of AD and explore the effect of TSG on the pathology of AD, thus providing molecular theoretical basis for the new drug creation of AD in clinical treatment. Method: (1) constructing a microgene (mini-gene) containing 7,8 variable splicing of the exon APP (mini-gene). A design needle of exon 6-9 is designed. PCR was carried out and the amplified fragment was inserted into the eukaryotic expression vector PCI-neo and transfected into the HEK-293FT cell line to detect the expression product of the splice isomer. The human embryonic renal cell (HEK-293FT) and the mouse neuroblastoma cells (Neuro-2a, N2a) transfected with APP mini-gene (including exon 6-9, Part 6-9, part). Intron 6,7,8) to simulate the splicing of APP exon 7,8 in the body. (2) the expression of APP microgenes in human neuroblastoma cells (SH-SY5Y) and N2a cell lines. Specific RT-PCR and fluorescent real-time quantitative PCR primers for APP770, APP751, APP695 and total APP are designed. CR (qPCR) measured the expression of APP splice isomers or APP-KPI+. The expression of GSK3 beta in N2a cells was overexpressed and the effect of GSK3 beta on APP variable splicing was observed with RT-PCR and qPCR. (3) co expression of splicing factor ASF and kinase inhibitor beta in HEK-293FT cells The interaction between ASF and GSK3 beta was detected. In addition, HA-ASF, Myc-GSK3 beta was transfected into HEK-293FT cells, ASF was then immunized, and phosphorylated specific antibodies were used to detect the degree of phosphorylation of ASF in the immunoprecipitation. ASF, GSK3 beta was separately or co transfected to He La fine cell, and the fluorescence two was stained to observe ASF and beta beta. (4) the SD (Sprague Dawley) rats were injected with insulin (Insulin) in the lateral ventricle. After 6 hours, RT-PCR and Western blot were used to detect the expression and activity of each signal molecule, and the PI3K-AKT pathway was detected, and the APP-KPI+ level was observed for 12 months in the TSG (50mg/kg/day) treatment of.APP/PS1 transgenic mice. The changes in the level of M RNA and protein in the rat brain were observed. Results: (1) APP mini-gene was expressed in HEK-293FT and N2a cell lines, and the results of RT-PCR showed three bands, which were APP770, APP751 and APP695. respectively. The expression of APP-KPI+ (770 and 751) was richer than that of APP-KPI- (695). Therefore, the microgene was studied. GSK-3 beta promoted the expression of APP microgene product APP-KPI+ in N2a cells, and Si GSK3 beta inhibited the expression of APP-KPI+; GSK3 beta inhibitor Li Cl treated SH-SY5Y cells at different concentrations, and the endogenous APP-KPI expression decreased with the increase of concentration. (2) all kinds of splicing factors were co transfected with miniature genes. Cells, in which ASF has the greatest influence on the expression of APP-KPI+, is the most important APP variable splicing regulator.GSK3 beta can be immunized by ASF, and there is a physiological interaction, and the immunocallocating experiment shows that ASF and GSK3 beta have good intracellular co localization. The kinase GSK3 beta can increase the intracellular splicing factor ASF serine phosphorylation However, if the cells that CO expressed ASF and GSK3 beta were pretreated with TSG for 48 h, the level of serine phosphorylation on ASF decreased. (3) TSG inhibited the expression of APP-KPI+ in HEK-293FT and SH-SY5Y cells, and found that the endogenous AKT-GSK3 beta signaling pathway of SH-SY5Y cells could be activated by TSG, and the level of phosphorylation was significantly increased. There was no significant change in the expression of AKT and GSK3 beta. The AKT-GSK3 beta pathway was activated after the injection of PI3K-AKT-GSK3 beta activator Insulin in the lateral ventricle of the rat. Meanwhile, the expression level of APP-KPI+ was reduced to P0.05. (4) compared with the control group (5 month old APP/PS1 transgenic mice were administered for TSG (50mg/kg/da) (50mg/kg/da). Y) 12 months later, the level of phosphorylation of AKT and GSK3 beta in the brain increased and the expression level of APP-KPI+ decreased. Conclusion: this experiment successfully constructed a microgene.TSG that studies APP-KPI expression can activate the AKT-GSK3 beta pathway in the brain of neural cells and APP/PS1 transgenic mice; GSK3 beta can phosphorylate factor ASF, thus inhibiting the ability to promote APP-KPI+ generation. Force; feeding TSG for a long time in the body can reduce APP-KPI+ expression and prevent Ab deposition. The results suggest that TSG may regulate the APP alterable splicing by activating the AKT-GSK3 beta signaling pathway, thus improving the pathological changes of Ab. The second part: the effect of EGCG on Dyrk1A regulated APP variable splicing: one of the pathological features of AD is the senile plaque formed by Ab deposition. The previous study found that 1A (Dual specificity tyrosine-phosphorylation-regulated kinase1A, Dyrk1A) is an important protein kinase in the brain, its activity is elevated in the AD brain and Dyrk1A is a potential gene variable splicing regulator, which has been shown to mediate microtubule related proteins. The variable splicing regulation of exon 10 of tau gene is regulated, but there has not been a report on the study of Dyrk1A's alterable splicing of APP. Epigallocatechin -3- gallate (epigallocatechin-gallate, EGCG) is a natural green tea extract with small toxicity. The purpose of this study was to study the effect of Dyrk1A on the variable splicing of APP microgenes and endogenous APP genes; Intracellular and whole animals inhibited Dyrk A activity, studied the expression changes of APP-KPI and the learning and memory behavior changes in Ts65Dn model mice. Methods: (1) transfection of Dyrk1A eukaryotic expression plasmid or non enzyme active mutant (dominant negative) Dyrk1ADN to N2a cells, Real-time PCR detected the effect of Dyrk1A on the expression amount. (2) culture The density of 5Y cells was about 80%, and the different doses of Dyrk1A inhibitor camel subtraction (Harmine) were added to the basic culture medium of DMEM/F12. After 24h, the total RNA was extracted from the cells, and the variable splicing of APP was analyzed by RT-PCR. The APP770 expression plasmid was constructed and transfected to HEK-293FT cells, and the expression of APP was changed by Western blot. The effect of preparation on the expression of APP-KPI+. (3) inhibition of Dyrk1A activity on behavioral changes and molecular biology in Ts65Dn model rats: the diet of Dyrk1A inhibitor EGCG in mice was given after birth. After 3 months of feeding, the changes of APP alterable splicing in the brain of the feeding and control mice and the changes of learning and memory behavior were observed. Results: (1) the transfection of Dyrk1A and inactive Dyrk1A to N2a cells showed that Dyrk1A significantly promoted APP-KPI expression, and there was no significant difference in the above changes. (2) the Dyrk1A inhibitor Harmine acted on SH-SY5Y cells in different concentrations, and could inhibit APP variable splicing, and the APP-KPI+ expression of the expression product presented Harmine concentration. The dependence was reduced. (3) the Dyrk1A mutants of the deletion of the Dyrk1A deletion mutants to the SH-SY5Y cells were observed to be compared with the total length of the missing C- terminal sequence. The statistical analysis of APP-KPI expression.RT-PCR and Western blot at different degrees suggested that the Dyrk1A missing mutants have greater influence on APP variable splicing than the wild type. (4) behavior Study changes: Ts65Dn model mice and normal control rats, spatial learning and memory ability significantly reduced the ability of.EGCG to reduce the level of APP-KPI+ in the brain of Ts65Dn mice, while the impaired spatial learning and memory ability improved. Conclusion: Dyrk1A regulates APP alterable splicing, Dyrk1A promotes APP-KPI+ production and inhibits its activity in APP-KPI+ expression. Reduction of Ts65Dn EGCG diet can inhibit Dyrk1A activity, decrease the expression of APP-KPI in brain, and improve learning and memory behavior changes.

【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R96

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